Proofreading is the primary guardian of DNA polymerase fidelity. Eukaryotes have two proofreading polymerases, Pol 5 and Pol e,that are required for faithful chromosomal DNA replication. To determine the biologic significance of proofreading, we made "knock-in" mice with inactivating point mutations in the exonuclease (exo) domains of these essential polymerases. Here we propose to characterize our new Pol eexo~ mutant line, together with Pol 8 exo" and mismatch repair (MMR) mutants, to delineate pathways that govern faithful DNA synthesis and cancer avoidance in mammals. The overall obective of this project is to determine the biologic functions of Pol e proofreading. The specific aims are: 1) characterize the survival and cancer phenotypes of Pol eexo" mice, 2) characterize the mutator phenotype of Pol e exo" cells and tissues, 3) determine the functional relationship of mammalian Pol 6 and Pol e proofreading, and 4) determine the cooperative roles of Pol eproofreading and MMR in mutation and cancer avoidance. Together, these studies will reveal the significance of Pol eproofreading to mammalian biology, DNA replication fidelity and cancer. These studies lay the groundwork for characterizing human polymorphisms that map to the proofreading domain of Pol e, and provide tools for developing novel strategies that exploit replication error-catastrophe for anti-tumor therapy.